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American-Eu ras ian Jo u rnal of Sus t ain ab le A g ricu ltu re, 3(2): 244-252, 2009
ISSN 1995-0748
© 2009, A merican Eu ra s ia n Net wo rk fo r Scien t ific In fo rma t ion
T his is a refereed jou rnal an d all articles are professionall y screen ed and rev iewed
ORIGIN AL AR TICLES
244
Corre spondin g Author: Dr. Savita Chaurasia, A s sistant Professor, Department of Biot echnology, CET,IILM Academy
of Higher Learning, 18, K nowledge Park II, Greater Noida-201306, G.B. Nagar, U.P. India.
Ph: +911202320056 Fax: +911202320058
E-mail: drsav16@rediffmail.com
Anti-inflammatory and Antioxidant Activity of Strychnos nux vomica Linn.
Savita Chaurasia
Department of Biotechnology, College of Engineering and Technology, IILM Academy of Higher Learning,
17,18 Knowledge Park II, Greater Noida-201306, Uttar Pradesh, India.
Sav ita Ch a u r a s ia, A n ti-inflammato ry & A ntioxidan t A ctivity o f Strychnos Nux Vomica Linn , Am.-
Eu ra sian J. Su stai n. Agric ., 3(2 ): 24 4 -252 , 200 9
ABS TRACT
Strychnos nux-vomica Linn. (Loganiaceae) is an evergreen tree native to Southeas t Asia. In traditional
med ic in al s y s tem see d s , ba rk an d le aves ha v e be e n u s ed in a v a rie ty of dis eas es . See d s a re wide ly u s e d in
trea t ment o f Ecze ma, rh eumat is m, p a ra ly s is , a s t hma , dia b e tes and piles e t c. In the p res ent s tu d y A n ti-
inflammatory and Antioxidant activities of the a lcohol extract of the Strychnos nux vomica was assayed in
Cha rles Fost er albino rat s. Ac ute a nd chro nic inflammation mod els were u sed to ev aluate th e anti-inflammato ry
activity (in vivo). In acute model carrageenan was used to induce inflammat io n in rat hind paw and cotton
pelle t-induced granuloma method was used for chronic inflammation model. The antioxidant property was
as s ess e d o n e n zymat ic an d no n-enzymatic models of lipid p ero xidation ind uced b y Fe -ADP (1.6 mM-62 µM )
3+
4
an d F eSO (0.5mM ) re s p e ctiv e ly (in vitro). Degree of lipid peroxidation was measured TBARS estimation. Level
of reduced glutathione (GSH) was estimated by the method of Ellman (1959). In order to evaluate
an tihepatotoxic act ivity of S. nux vomica extract level of serum transaminases (SGOT and S GPT) was
measured by the method of Reitman and Frankel (1957). The extract (50–200 mgkg b.w., p.o.) exhibited dose
-1
and time depend ent significant inhibition on both the models of inflammation. At a dos e of 200 mgkg b.w.
-1
fo r 7,15 a nd 30 d a y s 40% ,72% a nd 95% in h ib ition in o e d ema format io n (a c ute mo d e l) wa s f o u n d re s p e ctiv e ly .
In the c h r o n ic mo d e l at a d o s e o f 200 mgkg b .w . fo r 7 an d 21 day s there w as 24.75% and 58% in h ibitio n
-1
in co t t on pelle t granuloma re s p e c tively . T h e extract als o inhibite d b o th th e mo d els o f lipid p e ro xidat io n in a
50 4
dos e dep e n d ent ma n n er. ED was fo u n d to b e 1 4 9 µ g / ml a n d 85µg / ml o n Fe SO and Fe -A DP models o f lip id
3+
4
peroxidation respectively. It significantly inhibited aerobic as well as FeSO induced depletion of GSH in time
an d d o s e de p e n d ent ma n n er. Oral t re a tmen t o f d ru g u p t o 200 m g k g b .w . fo r 30 d ays d id not s how a n y ris e
-1
in serum transaminas es (SGOT and SGPT). The results obtained in this study indicated that the ethanol extract
of Strychnos nux vomica p osses s potent anti-inflammatory an d a ntioxidan t p roperty with n o d et ectable ad v ers e
effect. The se resu lts con firm the us e of S. nux vomica tradit ionally for the treatment of rheumatism and other
inflammatory conditions.
Key Wor ds: Strychnos nux vomica, A n ti-inflammat o ry , A n tioxid ant, Lip id p e ro xida t io n , Gluta t h ione
Introduction
It is now well established that free radicals have been implicated in a vast number of diseases , rangin g
fro m c ance r, th ro u g h a u toimmun e c o nditio n s , to ac u t e an d c h ro n ic inflamma t o ry d is e as e inc lu d in g rh e u ma t o id
a rt hritis (Halliwell, 1987, Halliwell and Gutteridge 1999; W in row, et al ., 1993; M ah ajan and Tand on, 2004).
This has led to increased interest amongst the researchers globally to evaluate role of antioxidan t t h erap y in
inflammato ry dis ea s es . In s pite of the discov ery of several newer ag en ts , the search for better anti-inflammatory
drugs continues becaus e they have many known side effects and n o n e of them is suitable for prolonged use.
The side effects of the anti-inflammatory drugs are one of t h e majo r p roblems in developing medicine today.
Therefore, development of new and more powerful drugs with fewer side effects is needed.
245
Am.-Eurasian J. Sustain. Agric., 3(2): 244-252, 2009
Natural products have long been recognized as an important source of therapeutically effective medicines.
Large numbers of h erb al drug s a re in us e for the treatmen t of arthritis b y A y urvedic and Siddh a p rac titione rs.
Ayu rveda recommen ds the use of Stychnos nux vomica Linn . in p u rified fo r m s i n c e t i me immemorial in
treatment of various diseases. Seeds are bitter, insecticidal, aphrodisiac, appetizer, tonic, antihelmintic,
feb rifuge, emmen ag ogu e, p urgative , stimulan t and s to mach ic (W arrier et al., 1996). They are useful in anaemia,
asthma, bronchitis, constipation, diabetes , ins omnia, cardiopalmus, skin diseases, paralysis and weakness of
limb s . Seeds a re a lso u s ed fo r nerv ou s d isord ers (Jain & De Filipps, 1991). It is very e ffec tiv e in ch icke n p o x
fever. It is a tribal remed y fo r snake bite (Murthy et al, 1986). It is wid e ly u s ed in trea t men t o f e c zema
(M a s lma n i et al., 1979 and 1981) and rheumatis m (Choudhury, 1977; Sen et al, 1983; Shukla et al ., 1985).
Different formulations of this plant product are o n the market, for treatment of rheumatoid arthritis and other
metabolic ailments (Thakur et a l., 1989; Chaurasia e t al., 1995).
Ant ilipid p e ro xid a tive prope rty of Strychnos nux vomica alcohol extract has been reported on cumene
hydro p eroxide (T rip ath i a n d Ch a u ras ia, 1996a) and ferro us s u lp h ate (T rip a t hi an d Cha u ra s ia , 1996b ) in d u c e d
models of lip id peroxidatio n . It a ls o p o s s es s e s s ig n ific a n t met al ch e latio n p rop e rt y a n d ch e la ted b o t h fo rms of
iro n (Fe2+ and F e 3 + ) . D u e t o lack of re d o x be h avoiur it d o e s n o t act as pro o xida n t wit h tran s ition me t al ion s
(Tripathi and Chau ras ia, 2000).
The present study was undertaken to screen the relationship between anti-inflammatory and antioxidant
activity o f S. nux vomica seed extract . Th e ant i-inflammatory act iv ity of Strychnos nux vomica Linn. extract
was studied on acute and chronic phases of inflammation using carrageenan induced paw edema (Winter
et al ., 1962) and the cotton pellet granuloma test (Bailey, 1988), respectively. The efforts has been made to
exp lain th e mech a n is m o f ac tion b y s tud y in g a n tioxidant p ro p e rt y o n enzymat ic an d n o n e n zy mat ic mo dels of
4
lipid peroxidation induced by Fe -ADP (1.6 mM-62 µM) and FeSO (0.5mM ) respectively accompanied by
3+
mea s u ring red u c e d gluta t h ione le v e l under n o r m a l a n d t o xic conditio n . In order to e v a luat e antih e p ato t o xic
activity o f S. nux vomica extract, level of serum trans aminases (SGOT and SGPT) was measured.
Mat e r i al s an d meth ods
Chemicals
Th io b a rb it u ric acid (T BA ), t ric h lo ro a cet ic ac id ( T C A ) , f e r r ic chlorid e, ferro u s s ulphat e and a c etic a cid
we re p u rc hased fro m Central D ru g Ho u s e Pv t. Ltd. 1,1,3,3-t e tra et h o xy p ropan e ( T E P), re d u ced g lu t ath io n e
(GSH), Adenosine di phosphate (ADP), 5,5-dithio-bis(2-nitro benzoic acid)(DTNB) and carrageen an w ere of
Sigma Chemical Co., Louis, Mo. USA. All other chemicals were of analytical grade.
Plant Material
S. nux vomica seeds were purchased from the A yurvedic Pharmacy, Institute of M edical Sciences, Banaras
Hindu Univers ity. Their authenticity was verified on pharmacognostic parameters and with the direct
compariso n with the sample preserved in the department of Dravy aguna, IMS, BHU (Vouch er No. 180). For
its purification, an indigenous method, as described in Ayurvedic text (Bhanu and Vasudevan, 1986) was used
as des cribed ea rlier (Tripathi and Cha uras ia, 1996a).
Preparation of alcohol extract
Dried purified s eeds were powdered and exhaustively extracted with ethanol using soxhlet extracto r for
48 ho urs. T he res u lting extract was dis tilled u nder reduced pre s sure in a Buchi ty pe rotary ev apo rator.
Co n c e n trate d extra ct (res idue) wa s t ra ns fe rre d t o a va c u u m des icca t o r and dried u n t il cons tan t weig h t w a s
at t ained . Th e y ield o f s o lven t free e xtra ct w as 32.9% (w/ v ). Th e extra ct w a s c h ara cte rize d b y HP LC-fin g e rp rint
(Tripa t h i a n d Cha uras ia., 1996b). Th is extract was su s pende d in a d rug veh icle (Tween 80: water; 1:9) for a
known concentration (w/v) and was stored at 4 C until further use.
0
Animals
Inbred A lb ino rat s o f Charles Foster strain (100 –150 g) of either sex were used for the pharmacological
ac t iv ities . They were kep t in p o ly pro p y lene c ages at 25 ± 2° C, with re la tive h u mid i t y 4 5 -55% un d e r 12h light
and dark cycles. All the animals were acclimatized to the laboratory conditions for a week before use. They
were fed with stan dard animal feed (Hindu s ta n Lever, Mu mbai, India.) and wa ter ad li bitum. The animals were
not fed for 12 hours before experiment.
246
Am.-Eurasian J. Sustain. Agric., 3(2): 244-252, 2009
Carrageenan induced paw oed ema
Th e ra ts were divided in to 5 grou p s (n = 6). Group I s erve d as co ntro l, which re ce ive d dru g ve hicle 10
mlkg b.w, p.o. (tween 80:water; 1:9), group II-V were pre treate d wit h S. n u x vomica extra ct (50, 100,150
-1
and 200 mg kg b.w, p.o.) as per protocol. Paw oedema was induced by injecting 0.1ml of (1%, w/v)
-1
carrageenan in ph ysiological s aline into th e sub plantar tis s ues of the left hind paw o f e a c h ra t (W inter et al.,
1962). Animals were pretreated with different doses of drug for 7,15 and 30 days prior to Carrageenan
ad minist ration. The paw v o lu me wa s mea s ure d every ho ur till 3 h after c arrageen an injection b y t he mercu ry
dis placement meth od u s ing a p leth y smograp h . The percen tage inh ibition o f paw v olume in d rug treat ed grou p
was compared with the control group. The anti-inflammatory a ctiv it y is expressed as the average percent
inhibitio n o f o e d ema in e ach g rou p , wh ich is calc u late d acc o rd in g t o the g e n eral formu la:
% in h ib ition = (Vc - Vt ) ´ 100/Vc, wh ere Vt and Vc represent the average oede ma volume of r a t s t rea t e d
wit h d ru g and co n t ro l, res pec t iv ely.
Cotton pellet granuloma
Th e ra t s were d iv id e d into 5 groups (n = 18). Gro u p I s e rv e d as control, wh ic h r e c e iv e d dru g veh ic le
10mlkg b.w, p.o. (tween 80:water; 1:9), group II-V w ere pre treated with S. nux vomica extract (50, 100,150
-1
an d 200 mgkg b.w, p .o.) as pe r pro to col. All fiv e g roup s were sub divided into 3 sub grou ps (SG) (n=6).
-1
Subg rou p 1 (SG1) was not p retreat ed with an y dru g. SG2 an d SG3 were pretreated with different do s es o f drug
for 7 and 21 days respectively. After completion of drug schedule, dry sterilized cotton pellets (10 ± 0.5 mg )
were implanted subcutaneously into both sides of the groin region of each rat. Different drugs were continued
fo r n e xt 7 d a y s . T h e pelle t s w e r e t a ken o u t o n 8 day , was h ed an d d rie d at 60 C for 24 h r. Th e g ra n u lo ma
th o
weig ht o btained fro m co ntrol and t rea te d gro u p s w ere used to ca lcu late p erc en tage inhib ition (Bailey, 1988,
Mukh opadh yay an d Lahiri, 1992).
Activity of serum transaminases
Different dos e s (50–200 mgkg b.w, p . o .) o f S. nux vomica extract were given orally for 30 days. After
-1
completion of drug schedule, blood was collected from brachial artery . S erum was separated and stored
immediately at -20 C. Serum glutamic oxaloacetic transaminas e (SGOT) and serum glutamic pyruvic
o
transaminase (SGPT) were determined using commercial kit (J. Mitra & Co. Pvt. Ltd., New Delhi) based on
t h e meth od o f Reitman an d Fran kel (1957). Dinitro ph en yl hydrazine us e d as s ub s tra te an d a b s o rb a n c e w a s
record ed at 520 nm ag ai ns t dis tilled wate r. Units of SGOT and SGPT were determined from the stand ard
curve.
Preparation of rat liver homogenate
Rats were fixed on the operation table by ventral side up and dissected. Liver was pe rfus ed with normal
saline throu gh h ep at ic po rta l vein. Liver was harv e s t e d a n d it s lobe s were briefly d ried be tween filter pap ers
an d w ere th in c u t with a h e a vy-d u ty bla d e . T hese s mall pie c es we re then t ra n s fe rred t o t he g la s s Teflon
homogen izin g tube t o p re p are homog e n a te (10% , w/v ) in P h o s p hat e b u ffe r s aline (P BS) (p H 7.4) in c o ld
condition. It was centrifu g ed at 3000 rpm for 10 minutes. The supernatant was finally sus pended in PBS to
contain approximately 0.8-1.5 mg protein in 0.1 ml o f s u s p ens ion, which was used to perform the in vitro
experiment.
Lipid peroxidation assay (TBARS)
The degree of lipid peroxidation was as s ayed by estimating the thiobarbituric acid-reactive substances
(TBARS) by using standard met hod (Ohkawa et al., 1979) with slight modifications (Pandey et al., 1994). 3
ml rat liver homogenate (5%) was taken in different 35 mm glass Petridishes. Different concentrations of p lant
extrac t a nd s t an d ard an tioxidan ts (as per p roto co l) we r e p r e in c u b at ed with h omog en ate for 10 min a t 37 C.
o
After incub ation lipid pe roxidation was ind u ce d enzymat ically a n d n o nen zymatically by a dding Fe3 -A DP (1.6
+
4
mM -62 µM ) a n d Fe SO (0.5mM ) res p ect iv ely. Pe trid is h e s w e r e further inc u b a ted for 30 min . 100 µl o f
incub ation mixture (5% homogena te in PBS, pH 7.4) was tra nsfe rred to a tub e c onta in in g 1 .5 ml 10% trichloro
acetic acid. After 10 minutes, tubes were centrifuged at 5000 rpm for 10 minutes. Supern a ta n t was mixed with
1.5 ml TBA (0.67% aqu eo u s TBA in 5 0% a ce tic a cid, 1:1). The mixture was kept in a b oilin g water bath fo r
30 min u tes. Tubes we re cooled a n d abs o rb a n ce was taken a t 535n m . T h e v a lu e s we re e v aluat ed on th e b as is
of a stand ard cu rve by u s ing 1, 1, 3, 3-tetra etho xy p r o p a n e (T EP). Pro te in was es timated by th e st an dard
method (Lowry et al, 1951).
247
Am.-Eurasian J. Sustain. Agric., 3(2): 244-252, 2009
Reduced glutath ione a ssay (GSH)
Reduced glutathione was determined by the method of Ellman (1959), 3 ml of 10% rat liver homogenate
was taken in 35 mm Petridis hes. In control only buffer was added, whereas in e xpe rime ntal groups all the
4
ag e n t s s u c h as extra ct, FeSO , v itamin E a n d parabe n zo q u in o ne (PBQ ) were a d d ed in d iffe r e n t combinat io n s
as p er p ro toco l. 250ml in c ubat io n mixtu re was mixed wit h 0.5 ml p rec ip itat in g b u ffe r (5% T r ic h l o r o ac etic a cid
in 1mM et h y len e d iamin e tetra acetic acid (EDTA). The sample was centrifuged at 2000 rpm for 10 min., and
th e s u p e rn a tan t mixed wit h 2.5 m l o f 0 . 1 M phos p hate b u ffe r (p H 8.0). The c o lo u r was d evelo p e d by ad d in g
100 ìl 5,5-dithio bis (2-nitrobenzoic acid) (DTNB) (0.01%). Absorbance was determined at 412 nm. The
co ncen tra tion o f red uced gluta th ion e was ev aluated by using the s ta nd ard curve of reduced gluta th ion e (GSH).
Statistical evaluation
Results giv e n here are mean ± SD of six separate experiments. Level of significance has been calculated
by using Student’s t test
Res ults and dis cussion
Effect on carrageenan in duced rat p aw oedema
Int erp lantar injection of carrag eenan in rat s led t o a time-depen dent inc rease in p aw thickness (Fig u r e 1);
this inc rease was observed at 1 h and was maximal at 3 h after administration. However, carrageenan-induced
paw edema was significantly reduce d in all phases of inflammation in a dose and duration dependent manner
by treatment with S. nux vomica e xtra ct. Pretreatment with drug for 7 days, a maximum 40% inhibition in paw
oe dema fo rmat ion was ob s erve d at a dos e of 200 mgkg b .w, followed b y 3 5% , 21 % a n d 11 % with 150, 100,
-1
50 mg kg b .w res p ect iv ely. On in c re asing th e d u ra tion o f drug trea t ment, t h e e xte n t of oede ma fo rma tion
-1
dec re a s e d s ig n ific antly . A t a d o s e o f 200 mg kg b .w. fo r 7,15 a n d 30 da y s 40% ,72% a n d 95 % in h i b i t io n in
-1
oedema formation was found respectively. It shows that S. nux vomica is effect iv e to c h e c k t h e i n fla mmatio n
on long term use.
Fig. 1: Effect of S. nux vomica on ca rrag eena n in d u c e d ra t p aw edema (d os e an d du ration resp o ns e ).
Measurements are made at 3 hours . Each v alue rep rese nt mean ± SD (n=6).
Effect on cotton pellet granuloma
Th e effects of S. nux vomica extract on the proliferative phase of inflammation are shown in Table 1. A
significant red uc tion in the weight of cotton pellets was o b s erv ed with an imals p ret reated with ext ract for 7
and 21 days (SG2 and SG3) in comparison with control rats . Res p onse was in a dose and time dependent
manne r. In Sub g roup 1 (SG1) on ly 7% inhib ition in granu loma formation was o bserved with a do s e o f 50
mgkg which increased to 30% by increasing t h e do s e o f drug up to 200 mgkg . Antiinflammatory respons e
-1 -1
als o in creas e d with in c re as in g th e d u ra tion of dru g tre a t m e n t . A t a d o s e of 200 mgkg b.w in h ib itio n in
-1
gra nulo ma weigh t incre as e d fro m 30% to 87%.
248
Am.-Eurasian J. Sustain. Agric., 3(2): 244-252, 2009
Table 1: Effect of Strychnos nux vomica on co tto n pel let g ranulo ma.
Group Dose Sub group 1 Sub group 2 Sub group 3
(kg bw ) ------------------------------------------------------------------------------------------------------------------------------
-1
Cotton weight % Inhibiti-on Cotton weight % Inhibiti-on Cotton weight % Inhibiti-on
(mg) (mg) (mg)
Control 10 ml 30.07 ± 1.07 - 30.07 ± 1.07 - 30.07 ± 1.07 -
Strychnos 50 mg 28.66 ± 0.86 7.02 26.67 ± 0.71 16.94 23.74 ± 1.13 31.53
baa
nux vo mica 100 mg 27.03 ± 1.02 15.14 23.18 ± 0.84 34.32 19. 43 ± 0.91 53.01
aaa
150 mg 25.65 ± 0.69 22.02 20.04 ± 0.43 49.97 15. 97 ± 0.82 70.25
aaa
200 mg 24.02 ± 0.90 30.14 17.79 ± 1.08 61.18 12. 60 ± 0.72 87.04
aaa
Each v alue repres ent m ean ± S D (n= 6).
St atis tical com paris on wi th co ntro l group, whi ch received onl y drug vehi cle (tween 80: water, 1:9 ).
P value : a < 0.001; b < 0.05
SG1: Animals were not pretreated with any drug. After implantation of cotton pellets, drug was given orally for seven days.
SG2 and SG3: Animals were pret re at ed wi t h dr u g for 7 and 21 days respectively and then cotton pellets were im planted and drug
administration was continued for next seven days.
Effect on serum transaminases
Res u lts (T able 2) cle a rly in d icat e th a t the re was n o s ig n ifican t c h a n g e in SGOT an d SGPT ac t iv itie s in
comparison to control. Thus it could be inferred that S. nux vomica up t o a d ose o f 200 mgkg body weight
-1
is totally safe when given to normal rats for a long duration of 30 days.
Table 2: Effect of Strychnos nux vomica on serum transam inases.
Group Dose SGPT (IU/ml) S GOT (IU/ml)
------------------------------------------------------- ------------------------------------------------------
(kg bw) 15 days 30 days 15 days 30 days
-1
Control 10 ml 33.22 ± 6.57 32.27 ± 5.69 76.39 ± 5.01 76.91 ± 5.82
Drug vehicle 10 ml 32.11 ± 4.67 27.99 ± 4.88 74.97 ± 4.01 74.54 ± 3.88
bbbb
Strychnos 50 mg 30.91 ± 3.03 30.37 ± 6.24 73.37 ± 4.92 73.13 ± 5.16
bbbb
nux vo mica 100 m g 32. 63 ± 3.98 29.30 ± 3.92 75.61 ± 3.67 75.41 ± 4.90
bbbb
150 mg 32.29 ± 4.77 28.67 ± 4.86 75.92 ± 4.03 74.69 ± 3.82
bbbb
200 mg 33.97 ± 5.31 28.43 ± 5.01 75.21 ± 3.92 74.08 ± 4.98
bbbb
Each v alue repres ent m ean ± S D (n= 6).
St atis tical com paris on w ith cont rol group, which receiv ed on ly, disti lled water.
P v alue: b = NS (not s ign ificant)
Protective effect of S. nux vomica on lipid peroxidation
4
Strychnos nux vomica extract showed s ig n ificant reduction in lipid peroxidation induced by FeSO and
Fe3 -ADP complex in a dose dep e nden t manner (Table 3). Under similar experimental conditions result was
+
co mpared with well known antioxidants Vitamin E and Parabenzoquinone (PBQ). Vitamin E and PBQ in
50
increas ing con ce ntrat ion inhibited b oth t h e mo d e ls of lipid pe roxidation. ED for all th e t ree agen ts are
de termined by usin g d ose res p ons e cu rve (Table 4).
Table 3: Effect of Strychnos nux vomica on lipid peroxidation.
S. nux vomica (µg/ml) TB ARS (nmoles/100mg protein)
-----------------------------------------------------------------------------------------------------
4
FeSO Fe -ADP
3+
00 446.54 ± 6.41 477.82 ± 7.98
25 352.16 ± 7.52 328.33 ± 9.42
aa
50 315.33 ± 6.68 302.37 ± 8.65
aa
100 272.50 ± 6.59 266.56 ± 7.96
aa
200 235.16 ± 3.97 223.67 ± 8.31
aa
400 164.21 ± 3.94 149.24 ± 7.32
aa
800 073.94 ± 4.94 069.84 ± 5.67
aa
Each v alue repres ent m ean ± S D (n= 6).
Statistical comparison with control value, which was arrived by
4
adding 0.5mM FeSO and (1.6 mM-62 µM) Fe -ADP for 30 minutes.
3+
P value: a < 0.001
Table 4: Comparative study of S. nux vo mica with Vitamin E and parabenzoquinone
50
Antioxidant ED (µg/ml)
----------------------------------------------------------------------------------------
4
FeSO Fe -ADP
3+
S. nux vom ica 149 85
Vitamin E 58 12
Parabenzoquinone 35 10
T hese d ata are best represen tati ve of six s eparate experim ents .
249
Am.-Eurasian J. Sustain. Agric., 3(2): 244-252, 2009
Effect of S. nux vomica on reduced glutathione
Glutatathione in reduced form (GSH) is an important endogenous antioxidan t. The result in Figure 2
indicated th at in rat liver homogenate GSH undergo aerial oxidation an d t here is a g radu al decrease in GSH
4
content which reached the basal level in 50 minutes (control group). In presence of FeSO (3.0 mM) there was
a sha rp de pletion in GSH co ntent . S. nux vomica extract s ignifican tly reduced the rate o f oxid ation o f GS H
4
even in presence of FeSO . Under similar conditions, vitamin E and parabenzoquinone failed to maintain the
GSH content. Interestingly PBQ e n h ance d the rate of oxidation of GSH, whereas vitamin E neither prevented
no r en han ced GSH oxidation (Figu re 3).
Fig. 2: Effec t o f S. nux vomica o n reduced glutathione level in normal and FeSO4 induced rat liver
ho moge nate (Time kinetics ). Each v alu e re pres ent mean ± SD (n=6), p < 0.001.
Fig. 3: Co mpa rative s tud y of S . nux vo mica , vitamin E and parabenzoquinone on reduced glutathio n e lev el.
Each value rep res ent mean ± SD (n =6).
Discussion
Strychnos nux vomica wa s e v aluat e d fo r it s a n ti-inflammat o ry a ctiv i t y i n a c u t e and ch ro n ic mo d els .
Significant anti-inflammatory activity was observed in both carrageenan induced paw oedema and cotton pellet
granuloma models. The extract showed maximum inhibition of 95% in carrageenan induced p aw oed e ma a t
th e d o s e o f 200 mg kg b .w. when animals we re p re treat ed wit h dru g fo r 30 day s . Ca rra g e enan in d u c ed oe d e ma
-1
is commonly used as an experimental animal model for acute inflammation (Winter, 1962). The development
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Am.-Eurasian J. Sustain. Agric., 3(2): 244-252, 2009
of o ed ema in th e p aw of t h e ra t a fter inject ion of c arrageenan was d es c ribed by Vinegar et al . (1969) as a
biphasic event. The initial phase observed during the first hour is attributed to the release of his tamine and
serotonin, the second phase is due to the release of pros t ag land in-like substances The result of the present
study indicates that S. nux vomica extra ct s ho wed s ign ific ant s up p re s siv e ac t iv ity in b ot h phas es . Bas ed o n this,
it could be argued that the suppression of the first phase may be due to inhibition of th e release of early
med ia t o rs , s u ch as his tamin e and seroto n in , a n d t he a c tion in t h e s eco n d p h a s e may be e xpla in ed by blo ckin g
an y step fro m arach ido n ic acid release to p ro s ta gland in format ion b y catalys is of c yclo-o xyg en ase.
Ch ro n ic inflammat io n in clud e s a proliferat io n of fib ro b las ts an d the in filtrat io n of n eutroph ils and e xudat io n
(Sp e c tor, 1969; Swingle a n d Sh id e man, 1972). Thes e ce lls c an b e e i t h e r s p re ad o r in gran u lo ma fo rm.
Nonsteroidal anti-inflammatory drugs (NSAIDS) decrease the size of granuloma which results from cellular
reaction by inhibiting granulocyte infiltration/inflammation, preventing gene ra tion of collagen fibers and
suppressing mu copolysaccharides (Suleyman, 1999). The S. nux vomica extrac t s h o we d s ig n ific a n t an t i-
inflammat o ry a ctiv it y in cot t o n -p e llet in d uce d g ra n u lo ma an d t h u s fo u n d t o be e ffec t iv e in c h ro n ic
inflammatory conditions, which reflected its efficacy in inhibiting fibroblasts proliferation, synthesis of collagen
an d mu c o p o ly s a cch a rid e s d u rin g g ra n u lo m a tis s u e fo rmation . Th e re wa s n o ris e in serum tran s amina s e s in a ll
tested dos e of S. nux vomica up to 30 days (Table 2). This finding s u pports its non-toxic effect at the
therapeutic doses in rats.
Recent studies suggest that the inflammatory tis sue damages are due to the liberation of re act ive oxygen
spe cies fro m p h a g o c y t e s inv ading t he inflammation s ites (Conn er a nd Grisha m, 1996; W inrow, 1993; Parke
an d S a p o t a , 1 996). To inves tigate if the anti-inflammatory effect o f S. nux vomica could be also related to
an t io xida n t act iv it y , the e xtrac t wa s e v alua t ed on no n e n zy matic and en zy ma tic mo d e ls o f lip id p ero xida t ion
4
induc ed by Fe SO and Fe3 -A DP complex res pective ly in t he rat liver h o mo g e n a t e (B uch er et al., 1983;
+
Svingen et al., 1979; Hog eberg et al, 1975). The res ults (Table 3) clearly in d icate the dose dependent
pro te ctive res po n se o f S. nux vomica extract on b o th the mod e ls o f li p i d p e r oxiatio n (Table 3). Re sults were
co mpa rab le t o well kno wn an tioxidan ts (Table 4).
Glutathione is an important endogenous antioxidant, which plays an important role in protecting cells
ag ainst oxidat ive stress via g lutathione redox sy s te m. Tiss u e g lutathione de pletion s eems to b e res po nsible for
ind uction of lipid pero xida tio n (Meis te r an d And erson , 1983). S. nux vomica extract significantly suppressed
4
the depletion of GSH by aero b ic oxidation as well as in pres ence FeSO (Figure 2). This indicated that S. nux
vomica p ro v id e s a type o f n o n -e n zy ma tic re d u c ing ag e n t in th e s y s t e m w h ic h s p a re s t h e reduce d g lu tat h io n e
from undergoing oxidation.
Th e b ig g es t doubt, w h ich a n t ioxida n ts ra is e, is that o f s u ic id al oxidativ e s t re s s , in d u ced b y c ertain
antioxidants (Koshy et al., 2003; Cao et al., 1997; Offer et al., 2000). These antioxidants can act as pro-
oxid ants in certain conditions like pres ence of trans ition metals (Kosh y e t a l., 2003; Cao et a l., 1997) o r at
high c on cent ra tions (Offer et a l., 2000) an d c a n ca u s e t he ce ll to un d e rg o s e v e re o xida tive s tres s u ltimat ely
resulting in suicidal cell death . As S. nux vomica possess potent metal chelation property and lack the redox
property, it can not act as a prooxidant an d ca n be considered as a safe antioxidant (Tripathi and Chaurasia,
2000).
Alkaloid s are the main bioactive ingredients in S. nux vomica, 80% of which are strychnine and brucine,
as we ll a s their deriva t iv es s u ch as b ru cine N -o xide o r is o s try chnin e (Cai et al., 1990). The antioxidant and
metal che lation p ropert y o f strych nine and brucine h ave been report ed by Tripathi an d Cha ura s ia (2000). In
view of the recent animal s tudies strongly sugges ting anti-inflammatory role of alkaloids (Barbosa-Filh o et al.,
2006), the an ti-inflammatory activity o f S. nux vomica may be explained due to presence of alkaoids.
Conclusion
The results of this study show that Stychnos nux vomica Linn. ext ract has anti-inflammatory activity
against early phase (acute paw edema), late phase (cotto n pellet granuloma) of inflammation without any
deleterio u s side effects. The anti-inflammatory activity could be attributed to the presence of alkaloids, and
ot her relate d syn ergis tic co mpo nen ts with antioxidant an d meta l ch elation p rope rty .
Acknowledgement
A u thor is than kfu l to D epart me n t of M edicin a l Ch e mis try , In s tit u t e o f Med ic a l Sc ience s , Bana ra s H in du
University, Varanasi, India for extending research facilities.
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Am.-Eurasian J. Sustain. Agric., 3(2): 244-252, 2009
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